WO2014118810A1

WO2014118810A1 - Serum-free culture medium for cancer stem cells

Info

Publication number: WO2014118810A1
Application number: PCT/IT2013/000030
Authority: WO
Inventors: Cristina ZANINI
Original assignee: Euroclone Spa
Priority date: 2013-02-01
Filing date: 2013-02-01
Publication date: 2014-08-07
Also published as: EP2951291A1

Abstract

The present invention relates to a medium and a method for the culture of cancer stem cells.

Description

DESCRIPTION

SERUM-FREE CULTURE MEDIUM FOR CANCER STEM CELLS

The present invention relates to a medium for the culture and enrichment of cancer stem cells.

Prior art

Cancer stem cells (CSCs) , also called "cancer initiating cells", are one of the most current and controversial topics in cancer research. Even though, as of today, no "technical" definition exists able to univocally indicate which is the real cancer- initiating cell, a functional definition does exist. CSCs have two fundamental characteristics: the ability to self-renew and tumorogenic potential. The cancer stem cell is, in fact, a cell which is able to self- propagate in vitro (self-renew) giving rise both to other cancer stem cells and to more advanced cells in the line of differentiation (asymmetric division) .

According to this definition, interesting developments may be advanced for conventional treatment with a revisitation of the current basis of anticancer treatment strategies.

The normal cytotoxic agents used in clinics are designed to strike actively proliferating cells.

In most cases however this is not sufficient to eliminate the residual stem cells which, thanks to their reduced proliferative activity and/or the presence of proteins able to extrude the chemotherapeutic drugs from the cell, are not struck. Such resistance mechanisms implemented by the CSCs would explain the relapses of the disease.

The theory of cancer stem cells may therefore pose new paradigms in terms of treating neoplasias and open up new scenarios for making anti-cancer treatment more selective and efficacious.

Summary of the invention

The purpose of the present invention is therefore to provide a medium for preparing cancer stem cells. The cells thus obtained can be used in the field of research to perform studies aimed at understanding the mechanisms of neoplastic diseases starting and evolving and above all, for the study of the drug resistance of neoplastic diseases.

Object of the invention

According to a first object, the invention relates to a culture medium for cancer stem cells and its use to increase the stem cell characteristics of cancer stem cells .

A method is also described for obtaining cancer stem cells starting from cancer cells.

According to a further embodiment of the invention, the use of the medium to induce stem cell characteristics in cancer cells is described.

According to another object, the invention provide a kit for the preparation of cancer stem cells.

Description of the drawings

Figure 1 morphology of the Medullo Spheres generated

(A) at time 0 (MB), at step 1 (MBS (pi)) and at step 4 (MBS(p4)), which were counted for the first 10 steps

(B) ; the graphs in figure 2 show the data relative to the phenotypes searched for in the medullo spheres; the graphs in Figure 3 show the results of the evaluation of typical markers in the medullo spheres generated in relation to the adherence lines (B=DAOY; ^A=U /228 ; #=ONS-76) ; Figure 4 shows the morphological appearance of the medullo spheres; figure 5 shows the results of the colon spheres obtained starting from CT26 in adherence lines (A); at the first step (B) and at the third step (C) ; the graphs in Figure 6 show the results of the search for CD133 (A) and CK20 (B) in the colon spheres; figure 7 shows the results of the TUBO mammospheres obtained expanded in adhesion (E) at the first step (PI) at the second (P2) and at the third step (P3); the graphs in Figure 8 show the results of the verification of mammospheres for the markers Sca-1⁺ (A), usashi-1⁺ (B) , Oct-4⁺ (C) and ALDH⁺.

Detailed description of the invention

According to a first object, in the present invention a medium is described for the cultivation of cancer stem cells.

In particular, cells of any human cancer line may be used.

According to a preferred embodiment, cancer cells of the Central Nervous System are used, in particular, obtained from medulloblastoma, cancer cells of the colon and cancer cells of breast cancer.

Alternatively, cancer cells of glioblastoma, cancer cells of lung cancer, cancer cells of ovarian cancer and prostate cancer may be used.

As regards the formula of the medium of the invention, this comprises:

i) a base medium; and

ii) a supplement. In particular, the base medium is Dulbecco' s Modified Eagle's Medium-F12 DMEM-F12 (DMEM: Dulbecco & Freemen, 1959; F12: HAM, 1965), available commercially.

As regards the supplement to the base medium, this is composed of a mixture of components.

According to a particular embodiment of the invention, the medium is serum-free.

With reference instead, to the components of the supplement, these may be amino acids, growth factors and other components such as vitamins and their derivatives (such as salts and esters), salts, proteins, enzymes, fatty acids, sugars, phospholipids, alcohols, hormones, sterols, amino alcohols, compounds with an amine and diamine structure, antioxidants, oligopeptides, etc.

In particular, the amino acids may be chosen from essential and non-essential amino acids, such as, for example: L-glutamine or L-carnitine.

The growth factors, instead, may be the growth factor of human recombinant fibroblasts (FGF) or the growth factor of human recombinant epidermic growth (EGF) . Vitamins and their derivatives may be represented for example, by: biotin, vitamin E, vitamin E acetate, retinol, retinil acetate.

The sterols may be cholesterol. The hormones may be, for example: insulin, T3 (triiodothyronine), progesteron, corticosteroids.

The sugars may be for example, by: galactose, glucose. The linoleic acid and the linolenic acid may be representative of the fatty acids utilisable in the present invention.

Phospholipids are instead represented by: lecithin and phosphatidylcholine .

Compounds with an antioxidant activity may in addition be represented by reduced glutathione.

Salts such as sodium chloride or sodium selenite may be comprised in the medium of the present invention. The proteins may have an enzymatic activity or not, such as for example: albumin, preferably bovine, peroxide dismutase, catalase, transferrin.

Other compounds such as ethanolamine and putrescine may equally be included in the formulation of the invention .

Some of such constituents are present in commercially available solutions ready for use, such as B27 and N2 ( Invitrogen) , the qualitative compositions of which are shown in the following tables.

Table 1: B27

BIOTIN

L-CARNITINE

CHOLESTEROL

CORTICOSTEROIDS

ETHANOLAMINE D (+) -GALACTOSE

GLUTATHIONE

LECITHIN

LINOLEIC ACID

LINOLENIC ACID

PHOSPHATIDYLCHOLINE

PROGESTERON

PUTRESCINE

RETINOL

RETINIL ACETATE

SODIUM SELENITE

T3 (TRI-IODO-1-THYRONINE)

DL- -TOCOPHEROL

DL- -TOCOPHEROL ACETATE

BOVINE ALBUMIN

CATALASE

INSULIN

SUPEROXIDE DISMUTASE

TRANSFERRIN

pH=7.0-7.5

Table 2: N2

TRANSFERRIN INSULIN PROGESTERONE PUTRESCINE SODIUM SELENITE

In the supplement, the FGF and the EGF are present so as to each have a concentration of about 20 ng/ ml in the final medium.

In addition, the pH of the medium according to the invention is about 7.0 - 7.5.

According to a preferred embodiment of the invention, a specific mixture of some supplements has been identified which are dosed in specific concentrations; such mixture is capable of inducing stem cell characteristics in the cancer cells.

In particular, the preferred mixture has a composition indicated in Table 3: Table 3

TRANSFERRIN 5-15 mg/ml

INSULIN 5-10 mg/ml

PROGESTERON 20-40 ng/ml

PUTRESCINE 10 ng/ml -5 ug/ml

SODIUM SELENITE 5-10 ug/ml

For the preparation of the medium of the invention, a first step, step a) of preparing the supplement, mixing L-glutamine, mixtures of supplements (such as N2 or B27), FGF and EGF, is performed. In a subsequent step b) , an appropriate portion of the supplement is mixed with the base medium.

Preferably, the supplement is mixed in such a quantity as to represent 5% (v/v) of the total final medium obtained.

As regards the preparation, it is advisable to prepare the supplement and then to divide it into separate portions .

In order to extend the shelf-life of the supplement, it is preferable to store it at -20°C and to use it within one week after defrosting.

According to a second object of the invention, a method is described for enriching the stem cell characteristics of cancer cells.

The expression "enriching the stem cell characteristics" is understood to mean that the cells treated with the method according to the invention have acquired a more immature and undifferentiated appearance from a morphological point of view, as may be observed under the electron microscope (in this respect see Figure 4, in which aspects of the undifferentiation in the spheres are evident compared to the cells adhering to the medulloblastoma) .

In addition, phenotypic analysis has shown that the appearance and/or increase in stem cells typical markers .

For example, CD133, CD144, CD44, ALDH1, Nanog, Oct-4, nestin (for neural cells), β-catenin, Mushashi-1, Sca- 1⁺, SOX-2, etc...

Consequently it has been shown that following treatment with the medium of the invention the cancer cells de-differentiated, acquiring characteristics typical of cancer stem cells.

According to a preferred embodiment, the method of the invention thus makes it possible to obtain cancer stem cells starting from cancer cells.

In an even more preferred embodiment, medullo spheres were obtained from medulloblastoma, colon spheres from cancer of the colon, mammospheres from breast cancer. The method of the present invention comprises, in particular, the steps of placing in contact a preparation of cancer cells with the medium of the present invention for a sufficient period of time to achieve the staminalization thereof.

Such contact time may be comprised of 5-15 days and generally of 7-10 days using a special low-attachment plastic permitting the cells to grow into spheres. It was thus surprisingly discovered that the medium of the invention can be validly used for the purpose of inducing stem cell characteristics in cancer cells. According to a preferred embodiment, the medium may be used in particular for the enrichment of cancer stem cells .

According to another preferred embodiment, the medium may be used to de-differentiate (or undifferentiate) cancer stem cells, that is to say to make them lose those characteristics acquired during the differentiation process.

In particular, such de-differentiation is of a morphological type, such as can be observed under the electron microscope, and phenotypic.

In fact, it has been observed that the medium described in the present patent application is able to increase the expression of markers typical of stem cells, such as, for example: CD133, CD144, CD44, ALDH1, Nanog, Oct-4, nestin (for neural cells), β- catenin, ushashi-1, Sca-1⁺, SOX-2, etc... According to a further aspect, the medium described in the present invention may be used to obtain the growth into spheres (that is to say, in suspension) of cells which grow in adherence.

Again, the medium which the present patent application relates to makes it possible to obtain the serum-free growth in spheres of cells which need serum to grow. According to another object, the invention makes available a kit for the preparation of cancer stem cells .

In particular, such kit comprises:

the serum-free medium described above according to the present invention;

a solid support for a cell preparation.

With reference instead to the solid support, this is characterised by a low cellular adhesion thanks to the minimal absorption of the surface proteins.

In addition, this must be sterile or suitable for sterilising, biologically inert, non cytotoxic and not degradable over time.

A useful element for the purposes of the present invention may for example be an element, such as a plate made in so-called ultra-low attachment plastic.

EXPERIMENTAL PART

Materials and Methods For the purpose of obtaining spheres for the expansion and maintenance of "cancer stem cells", proceed as shown schematically below with reference to the cellular line B16 obtained from murine melanoma.

Defreezing of cells

• Bring the medium for the expansion of the B16, RP I 10% FS to room temperature.

• Defreeze a portion of cells in a bath with thermostat at 37 °C.

• Once defrozen, transfer the cells to a sterile falcon container and collect the remaining cells

• Add RPMI 10% FBS to volume

• Centrifuge the cellular suspension

• Discard the supernatant

• Re-suspend the pellet

Cell count

• Dilute the cellular suspension in PBS

• Colour the dilution with Trypan Blue and count 10 μΐ in Burker chamber

• Count the vital cells in the 2 diagonals and apply the following formula:

• [(diagonal 1 + diagonal 2)/2] * 2*105 = n° cells/ml

Cellular growth and sowing

• Plate the cells in one or more flasks • Place the flask in an incubator at 37°C

• Leave the cells overnight

• If necessary replace the culture medium

• Expand the cells for 1 or 2 weeks depending on the number of melano spheres to be obtained.

Changing -the medium

• Bring the culture medium and the PBS to room temperature .

• Aspirate and discard the medium from the flask

• Wash

• Add fresh FBS

• Replace the flask in an incubator at 37 °C

Cell step

• When the cells have merged, aspirate the medium

• Wash once

• Add Trypsin

• Incubate at 37°C

• Check that the cells have detached under the microscope

• Aspirate the cells

• Pellet in the centrifuge

• Discard the supernatant

• Re-suspend the cells

Spheres

• Detach the cells from the flasks. • Count the cells as in point (2) .

• Re-suspend the cells in a suitable medium for the formation and maintenance of the spheres.

• Put 5 ml of suspension of cells in a flask

• Replace the flask in an incubator

• The cells will grow not in adhesion forming melanospheres

Spheres step

• The spheres should be passed about every 7 days

• Collect the spheres

• Pellet the melanospheres in the centrifuge

• Discard the supernatant

• Re-suspend the pellet

• Incubate for 3 or 4 minutes at room temperature

• Wash with medium and/or PBS

• Pellet the mammospheres once again

• Discard the supernatant

• Re-suspend the pellets in the dedicated medium as in point (6)

• Put the melanospheres back into the flask as in point (6)

• Replace the flasks in an incubator at 37 °C

Characterisation by the cytofluorxmeter • At each step the spheres are collected, centrifuged, trypsinised, washed and recentrifuged for colouring in the cytofluorimeter

• Discard the supernatant

• Fix and seal the melanospheres

• Wash the spheres and re-centrifuge

• Discard the supernatant

• Re-suspend the melanospheres in the buffer

• Block with rabbit serum

• Wash the spheres and re-centrifuge

• Discard the supernatant

• Re-suspend the melanospheres in the buffer

• Colour the spheres fixed and sealed for the FACS with the primary antibody

• Incubate for 30 minutes at 4°C and in ice

• Wash the spheres with the buffer as above and centrifuge

• Discard the supernatant

• Colour the cells with the secondary antibody

• Incubate for 30 minutes at 4°C and in ice

• Wash the spheres with the buffer and centrifuge

• Re-suspend the melanospheres in the buffer and read off to the cytofluorimeter .

EXAMPLE 1

Cancer cells of the Central Nervous System The stabilised lines of medulloblastoma : DAOY, UW228 and ONS-76 were assayed for the generation of cancer stem cells using the medium of the present invention. Figure 1 shows the morphology of the medullo spheres thus generated, while the results of the count of such cells were continued for 10 steps (the results are shown in Figure 2) .

The presence of markers typical of stem cells was also verified in the medullo spheres thus generated. In particular, the presence of: CD133, Nanog, Oct-4, nestin and β-catenin was assessed. The results of the assay are shown in Figure 3 (A:CD133; B: β-catenin; C: nestin) .

In addition to the above, the morphological appearance of the medullo spheres obtained thanks to the use of the medium of the invention (MBS) was compared with that of the starting lines (MB) .

A much more immature morphological appearance was thus observed of the medullo spheres obtained according to the present invention.

Consequently, thanks to the medium of the invention an overall enrichment in stem cell characteristics as shown by both the morphological and phenotypic results was obtained.

EXAMPLE 2 Cancer cells of the colon

Cancer cells of the colon, human (HT29) and murine (CT26) were expanded in adherence with mediums containing the serum to verify the morphological characteristics and characteristics of proliferation and stability over time.

The results are shown in Figure 5.

The phenotype of the CT26 cells thus obtained and of the cells grown in adherence was characterised by means of a cytofluorimeter to verify the presence of the markers CD133 and CK20.

The results are shown in Figure 6 (A:CD133 and B: CK20) and show the ability of the CT 26 cells to grow as colon spheres maintaining positivity for CD133.

Consequently, an overall enrichment in stem cell characteristics as shown by the morphological and phenotypic evidence was obtained.

In addition, the findings of Chu P. et al (Mod. Pathol, 2000) and that is, that the positivity to CK20 is inverse to the loss of positivity for CD133⁺, was confirmed .

EXAMPLE 3

Cancer cells of breast cancer

Cancer cells of breast cancer (AG12) (TuBo) were expanded in D20 with mediums containing serum to verify the morphological characteristics and characteristics of proliferation and stability over time .

Mammospheres were obtained and the results for TUBO expanded cells (E) and after the first (P2) second (P2 and third steps (P3) are shown in Figure 7.

The presence of typical markers in steps 1,2 and 3 (respectively PI, P2 and P3) was verified by means of FACS.

The results are shown in the graphs in Figure 8 and show an enrichment in the markers of stem cell characteristics .

The formulation according to the invention has made it possible to generate positive mammospheres for some of the stem cell markers up to the 7th step.

The numerous advantages offered by the present invention will be evident to a person skilled in the art from the above description of the medium for the cultivation of cancer stem cells.

For example, it will be possible to cultivate cancer stem cells starting from cancer cells, for example obtained from explants and biopsies.

The treatment of the preparations of cancer cells thereby de-differentiation to be induced and, thus the expression of the characteristics typical of cancer stem cells.

By analysing the phenotype of the cells thus obtained, the appearance or increase in the presence of markers characteristic of cancer stem cells was observed, confirming that de-differentiation had taken place. From a practical point of view, the medium can be prepared starting from known and commercially available components. It can also be stored at low temperature and thus be stored for a given period. A person skilled in the art may also prepare a batch to be divided into smaller portions, to be defrozen and used as needed.

Claims

1. Serum-free medium for the culture of cancer stem cells comprising:

- a base medium;

- a supplement.

2. The serum-free medium according to claim

1, wherein said base medium is Dulbecco's Modified Eagle's Medium-F12 DMEM-F12.

3. The serum-free medium according to claim

1 or 2, wherein said supplement comprises a mixture of transferrin, insulin, progesterone, putrescine and sodium selenite.

4. The serum-free medium according to claim

3, wherein the components are present in the following concentrations:

transferrin about 5-15 mg/ml insulin about 5-10 mg/ml progesteron about 20-40 ng/ml putrescine about 10 ng/ml -5 μg/ml sodium selenite about 5-10 μg/ml.

5. The serum-free medium according to any of the previous claims having a pH of about 7.0-7.5. The serum-free medium according to any of the claims 3 or 4, wherein said supplement further comprises one or more components chosen from among amino acids, growth factors, vitamins and their derivatives, salts, proteins, enzymes, fatty acids, sugars, phospholipids, alcohols, hormones, sterols, amino alcohols, compounds having an amine and diamine structure, antioxidants, oligopeptides.

The serum-free medium according to the previous claim, wherein the amino acids are one or more among essential and non-essential amino acids.

The serum-free medium according to the previous claim, wherein said amino acids are one or more among L-glutamine or L-carnitine.

The serum-free medium according to claim 6, wherein said growth factors are one or more among the growth factors of human recombinant fibroblasts (FGF) and the growth factor of human recombinant epidermic growth (EGF) . The serum-free medium according to claim 6, wherein said vitamins are one or more of biotin, vitamin E and its derivatives, retinol and its derivatives .

The serum-free medium according to claim 6, wherein the sterol is cholesterol .

The serum-free medium according to claim 6, wherein the hormones are one or more among T3 and corticosteroids. The serum-free medium according to claim 6, wherein the sugar is one or more among galactose and glucose.

The serum-free medium according to claim 6, wherein the fatty acid is one or more among linoleic acid and linolenic acid.

The serum-free medium according to claim 6, wherein said phospholipid is one or more among lecithin and phosphatidylcholine .

The serum-free medium according to claim 6, wherein said anti-oxidant is reduced glutathione. The serum-free medium according to claim 6, wherein said proteins are one or more among albumin, peroxide dismutase and catalase.

The serum-free medium according to claim 6, further comprising ethanolamine .

A method for the culture of cancer stem cells comprising the step of placing a preparation of cancer cells into contact with the serum-free medium according to any one of the claims 1 to 18.

The method according to the previous claim, wherein the contact between the preparation of cancer cells and the serum-free medium is protracted for a period of 5-15 days and preferably 7-10 days .

Use of the serum-free medium according to any one of the claims from 1 to 18 to enrich cancer stem cells.

Use of the serum-free medium according to any one of the claims from 1 to 18 for the morphological de- differentiation of cancer stem cells. Use of the serum-free medium according to any one of the claims from 1 to 18 to increase the expression of markers typical of stem cell characteristics in cancer cells.

Use of the serum-free medium according to the previous claim, wherein such markers are chosen from the group comprising: CD133, CD144, CD44, ALDH1, Nanog, Oct-4, nestin, β-catenin, Mushashi-1, Sca-1⁺, SOX-2.

Use of the serum-free medium according to the previous claim, wherein said markers are chosen from: CD133, ALDH1, Nanog, Oct-4, nestin, β-catenin, Mushashi-1, Sca-1+.

Use of the serum-free medium according to any one of the claims from 1 to 18 to obtain the growth in suspension of cancer stem cells.

Use of the serum-free medium according to any one of the claims from 1 to 18 to obtain the growth in spheres of cancer stem cells.

A kit for the culture of cancer stem cells comprising:

- the serum-free medium according to any one of the claims from 1 to 18,

- a solid support for a cell preparation .

The kit for the culture of cancer stem cells according to the previous claim, wherein said support is made from a material characterised by a low cellular adhesion.